Blood cultures: past , present and future

Dr Natalia Solomon MD, FRCPSC Medical Microbiologist

DynaLIFEDx Faculty/ Presenter Disclosure

 Faculty: Dr Natalia Solomon  Relationships with commercial interests:  Grants/Research Support: None  Speakers Bureau/Honoraria: None  Consulting Fees: None

 Other: Consultant for DynaLIFEDx Laboratories Disclosure of Commercial Support

 This program has received financial support from DynaLIFEDx in the form of an Educational Program.  This program has received in‐kind support from DynaLIFEDx in the form of logistical support • Potential for conflict(s} of interest:  Dr Natalia Solomon has received no honorarium and provides consulting services to DynaLIFEDx.  DynaLIFEDx provides laboratory services which will be discussed in this program. Mitigating Potential Bias

 DynaLIFEDx operates in accordance with Alberta Health Services, testing and solutions are a direct result of provincial standards. Case

 36 y.o. previously healthy female , developed nasal stuffiness, mild sore throat, and a cough 3 days ago.  Today she presented with fever up to 40°C , chills, progressively worsening cough with purulent sputum, and fatigue.  Based on a history and physical examination you are suspecting bacterial pneumonia and in addition to other investigations ordering blood cultures… Blood cultures: Volume

What is the recommended total volume of blood per culture? a) 5‐10 ml b) 20‐30 ml c) 40 ml d) 50 ml e) More is better Percentage increase for all pathogens recovered related to the volume of blood cultured

F. R. Cockerill III et al. Clin Infect Dis. 2004;38:1724-1730 Blood cultures: Volume

 The volume of blood that is obtained for each blood culture is the single most important variable in recovering microorganisms from patients with blood stream infections.  Recommended volume of blood to obtain from adults per culture is 20‐30 ml  Recommended volume of blood per bottle for adult patient is 8‐10 ml Checklist for blood cultures collection

 Timing of blood cultures and the optimum interval between successive blood cultures  Volume of blood (total and per bottle)  Number of sets (2‐3 sets per episode)  Separate blood cultures  Site  Clinical information Case: You received a phone call from the lab…

Scenario 1 Scenario 2

 Gram positive cocci in chains  Gram positive cocci in chains  1/3 vials  3/3 vials  After 26 hours of  After 10 hours of incubation incubation

 Culture : Streptococcus  Culture : Streptococcus sanguinis (VGS) pneumoniae Check list for blood culture results interpretation

 Organism  Time to detection  Total number of bottles vs number of positives  Aerobic vs anaerobic  Clinical diagnosis  Previous/following/other cultures available Blood cultures: Limitations

 Mycobacteria, filamentous and dimorphic fungi will not grow using routine BC method‐will require special media (MycoF/lytic vials, lysis‐centrifugation vials) and extended incubation  Coxiella burnetii, Chlamydia, Rickettsia, Tropheryma whipplei ‐ uniformly uncultivable, use serology or molecular methods  Bartonella, Legionella‐ can be isolated using lysis‐centrifugation vials and special culture protocols but more optimally diagnosed by serology or molecular methods  Blood volume and diagnostic yield  Time from sampling to incubation Blood cultures: Instrument Identification of the organisms‐past, present , and future

 Gram stain Identification of the organisms‐past and present

 Morphologic characteristics Identification of the organisms‐past

 Manual biochemical tests

 Culture‐based automated systems  Longer turnaround time  Expensive consumables  Priori knowledge of organism type Identification of the organisms‐ present

 MALDI‐TOF MS (Matrix Assisted Laser Desorption/ Ionization Time‐of‐Flight Mass Spectrometry) MALDI‐TOF MS : How it works

. The target slide is prepared and introduced to a high‐vacuum environment . A precise laser burst desorbs and ionizes the sample . A “cloud” of proteins is released and accelerated by an electric charge . The ionized analytes (proteins) enter a flight tube and are separated by their individual mass . A mass spectrum is generated and represents the number of ions hitting the detector at the end of the flight tube at a specific time (time of flight) . Generated mass spectrum compared against a database and software scores the relatedness of the test isolate’s spectrum to the known spectra stored in the system’s library MALDI‐TOF MS: Advantages

 Volume‐single or multiple isolates may be tested at a time, capable of analyzing thousands of samples per day  Decreased Turnaround Time, superior speed (the analytical turnaround time is ≤3 min per isolate)  Cost‐effectiveness  Accuracy ‐identifies organisms to the genus and species level. No interpretation required by technologist.  Database ‐ comprehensive collection of clinically relevant bacteria and fungi that can be customized and consistently updated MALDI‐TOF MS: Limitations

 Does not provide antimicrobial susceptibility results  Can not be routinely applied directly to clinical specimens ‐organism must first be cultivated  Uncultivable organisms cannot be identified  Database may be less comprehensive for esoteric organisms MALDI‐TOF : Future Developments

 Organism identification directly from clinical specimens  Specimen with relatively little human protein (blood, urine)  Requires high bacterial counts  Identification of organisms in mixed cultures  Requires extensive preparatory specimen processing  Would not provide antimicrobial susceptibility results Identification of the organisms: Molecular Methods

Molecular methods for the diagnosis of sepsis

Hybridization Amplification Non‐nucleic acid techniques techniques based techniques Identification of the organisms: Molecular Methods

 Hybridization  FISH

 Amplification  PCR  Multiplex PCR  Broad‐range PCR

 Non‐nucleic acid based  Spectrometry  Phage assays Diagnosis of sepsis independent of blood culture: Molecular Methods

 Applied directly on whole blood samples  No time‐consuming culture required  Include broad‐range and multiplex PCR  Most of them still used as a research tool  Clinical utility and benefits to be determined Molecular methods: Advantages

 Faster than conventional blood cultures

Method TAT after TAT after whole positive BC blood sample Conventional 18‐24h‐few days Culture growth needed • Hybridization 1.5‐3h 12‐24+h • Amplification 1‐8h 12‐24+h • Mass spectrometry 4‐6h 12‐24+h Directly from blood sample • Whole blood 6h amplification methods Molecular methods: Advantages (continue)

 May yield better sensitivity  Better detection in antimicrobial pre‐treated patients  May detect specific resistance profiles (e.g. mecA gene) Molecular methods: Limitations

 Critical hours after diagnosis are still lost  Pathogen panels may not include all relevant organisms  Detect DNAemia, not bacteremia, therefore, interpretation of positive result may be challenging (viable? contamination ? infection?)  A limited number of bacterial resistance genes can be routinely detected , however, those methods do not provide full antimicrobial susceptibility results for the detected pathogen Molecular methods: Limitations (continue)

 Microbial nucleic acid extraction required  Expensive (equipment, reagents, labor)  Resources ( dedicated space, trained personnel)  Data indicate that more rapid microbiological diagnosis does not ensure more rapid adequate antimicrobial therapy Antibiogram

 Antibiogram‐cumulative antimicrobial susceptibility test data summary  Generated by analysis of results on isolates from a particular institution(s) in a defined period of time (annually)  Reflects the percentage of first isolates (per patient) of a given species that is susceptible to each of the antimicrobial agents routinely tested  Should be used to guide empirical therapy decisions Antibiogram

 www.dynalifedx.com Rapid Antigen Detection Test (RADT) for Group A Streptococcal Pharyngitis: an update Group A Streptococcal Pharyngitis: RADT

 Developed in early 1980’s to provide rapid diagnosis of acute Streptococcal pharyngitis  Test identifies the presence of the carbohydrate antigen unique to Group A Streptococcus by an immunologic reaction  Sensitivity 70‐95%, specificity 95% Group A Streptococcal Pharyngitis: RADT

RADTs Sensitivity Specificity Comments Latex • Point of care test agglutination • Sensitivity increased with the Enzyme newer generation assays ( the immuniassay 70‐90% 95% most sensitive test being OIA) (EIA) • Takes about 15 min to complete • Optical Very simple to perform immunoassay (OIA) DNA probe 86‐94.8% 95‐100% • Employ molecular biology methods • Some can be a point of care tests Real‐time • Involve multiple steps, require PCR 93% 98% special equipment and batching • Takes 1.5‐2h to complete RADT : Principles

 RADT RADT : what is available today RADT : Advantages

 Rapid Diagnosis and Treatment  Results in approximately 5‐10 minutes  Patients can be tested and treated in the same office visit  Helps effectively manage antibiotic use  Easy to use  Convenient  Reasonable performance  Room temperature storage and long shelf life RADT : Limitations

 Qualitative  Unable to differentiate asymptomatic carriers from infections  False positive results can be due to cross reacting with other Streptococcus species such as Streptococcus anginosus group, heavy colonization with S.aureus  False negative may occur in patients with acute pharyngitis due to Group C and Group G Streptococci, with low bacterial load in the specimen (carriers, collection technic , prior antibiotics, etc.) Group A Streptococcal Pharyngitis: RADT

 In children and adolescents, negative test requires throat culture  In adults, routine back‐up cultures for negative results generally not recommended  Sensitivity to be determined by each practice/group  Sensitivity can be improved by applying clinical score RADT: Clinical Score

Criteria: Approach: 1.Tonsillar exudate or erythema 1. Strep Score 4 to 5: 2. Anterior cervical adenopathy  Treat with antibiotics 3. Cough absent 2. Strep Score 2 to 3:  Perform rapid antigen test 4. Fever present  Antigen test positive: Treat 5. Age: with antibiotics  Age 3 to 14 years: +1 point  Antigen test negative: Throat Culture  Age 15 to 45 years: 0 points 3. Strep Score 0 to 1:  Age over 45 years: ‐1 points  Provide Pharyngitis Symptomatic Treatment Thank you !